Among the techniques used for single molecule detection, wide-field optical microscopy offers the advantages of high throughput, multiple parameters and dynamic real-time monitoring. In the review, the wide-field optical microscopy apparatus, methods, detectable parameters, imaging probes, identifications criterion of single molecules, and applications in the fields of analytical and biophysics are summarized. Single molecule imaging is trending toward apparatus commercialization, operation simplicity, and process visualization with more accuracy in a wider and more complicated field. In the near future, the topics of single molecule detection will focus on quantitative analysis of biological sample, sub-diffraction-limit resolution, important biological mechanism, and characterization of nano-materials. Contents
1 Introduction
2 Apparatus for wide-field imaging
2.1 Epi-fluorescence microscope
2.2 Total internal reflection fluorescence microscope
2.3 Dark-field microscopy
3 Probes
3.1 Fluorescent probes
3.2 Scattering probes
4 Identification of single molecules and measurable parameters
4.1 Principles to identify single molecule
4.2 Parameters
5 Applications
5.1 Single-molecule quantification
5.2 Molecular behavior related to separation
5.3 Molecular interactions
5.4 Single molecule tracking
5.5 Single-molecule far-field nanoscopy
6 Outlook